Immunopathology of terminal complement activation and complement C5 blockade creating a pro-survival and organ-protective phenotype in trauma.

BACKGROUND AND PURPOSE: Traumatic haemorrhage (TH) is the leading cause of potentially preventable deaths that occur during the prehospital phase of care. No effective pharmacological therapeutics are available for critical TH patients yet. Here, we identify terminal complement activation (TCA) as a therapeutic target in combat casualties and evaluate the efficacy of a TCA inhibitor (nomacopan) on organ damage and survival in vivo. EXPERIMENTAL APPROACH: Complement activation products and cytokines were analysed in plasma from 54 combat casualties. The correlations between activated complement pathway(s) and the clinical outcomes in trauma patients were assessed. Nomacopan was administered to rats subjected to lethal TH (blast injury and haemorrhagic shock). Effects of nomacopan on TH were determined using survival rate, organ damage, physiological parameters, and laboratory profiles. KEY RESULTS: Early TCA was associated with systemic inflammatory responses and clinical outcomes in this trauma cohort. Lethal TH in the untreated rats induced early TCA that correlated with the severity of tissue damage and mortality. The addition of nomacopan to a damage-control resuscitation (DCR) protocol significantly inhibited TCA, decreased local and systemic inflammatory responses, improved haemodynamics and metabolism, attenuated tissue and organ damage, and increased survival. CONCLUSION AND IMPLICATIONS: Previous findings of our and other groups revealed that early TCA represents a rational therapeutic target for trauma patients. Nomacopan as a pro-survival and organ-protective drug, could emerge as a promising adjunct to DCR that may significantly reduce the morbidity and mortality in severe TH patients while awaiting transport to critical care facilities.

HMGB1 Inhibition to Ameliorate Organ Failure and Increase Survival in Trauma.

Several preclinical and clinical reports have demonstrated that levels of circulating high mobility group box 1 protein (HMGB1) are increased early after trauma and are associated with systemic inflammation and clinical outcomes. However, the mechanisms of the interaction between HMGB1 and inflammatory mediators that lead to the development of remote organ damage after trauma remain obscure. HMGB1 and inflammatory mediators were analyzed in plasma from 54 combat casualties, collected on admission to a military hospital in Iraq, and at 8 and 24 h after admission. In total, 45 (83%) of these patients had traumatic brain injury (TBI). Nine healthy volunteers were enrolled as controls. HMGB1 plasma levels were significantly increased in the first 8 h after admission, and were found to be associated with systemic inflammatory responses, injury severity score, and presence of TBI. These data provided the rationale for designing experiments in rats subjected to blast injury and hemorrhage, to explore the effect of HMGB1 inhibition by CX-01 (2-O, 3-O desulfated heparin). Animals were cannulated, then recovered for 5-7 days before blast injury in a shock tube and volume-controlled hemorrhage. Blast injury and hemorrhage induced an early increase in HMGB1 plasma levels along with severe tissue damage and high mortality. CX-01 inhibited systemic HMGB1 activity, decreased local and systemic inflammatory responses, significantly reduced tissue and organ damage, and tended to increase survival. These data suggest that CX-01 has potential as an adjuvant treatment for traumatic hemorrhage.

An Open-Globe Porcine Injury Platform for Assessing Therapeutics and Characterizing Biological Effects.

Open-globe injuries can result in permanent vision loss, partly due to extended delays between injury and medical intervention. Even with early intervention, the management of open-globe injuries remains a challenge for ophthalmologists, mostly due to inadequate or suboptimal current therapies. To aid in the development of novel therapeutics and track toxicological and pathophysiological changes, this article details an open-globe injury platform capable of inducing injuries in enucleated porcine eyes. The injury platform relies on a high-speed solenoid device to mimic explosive injury scenarios, allowing for large, complex injury shapes and sizes that are often observed in casualties and are more difficult to treat. The system can be implemented with precise computer control of the injury mechanism to allow for more complex setups. Also, the system can make use of real-time intraocular pressure measurement to track changes during injury induction and to assess therapeutic efficacy for restoring intraocular pressure and the integrity of the eye. These protocols will assist with implementation of the injury model in prospective laboratories seeking to develop therapeutics or studying biological changes that occur from this type of traumatic injury. Published 2020. U.S. Government. Basic Protocol 1: Preparing gelatin molds and porcine eye tissue Basic Protocol 2: Creating an open-globe injury using a solenoid device Alternate Protocol 1: Constructing a computer-controlled system for open-globe injury Alternate Protocol 2: Constructing a pressure measurement system for tracking intraocular pressure Support Protocol 1: Assessing ocular compliance in porcine eyes Support Protocol 2: Assessing outflow rate from the anterior chamber Support Protocol 3: Assessing burst pressure in porcine eyes.

Development and Characterization of a Benchtop Corneal Puncture Injury Model.

During recent military operations, eye-related injuries have risen in frequency due to increased use of explosive weaponry which often result in corneal puncture injuries. These have one of the poorest visual outcomes for wounded soldiers, often resulting in blindness due to the large variations in injury shape, size, and severity. As a result, improved therapeutics are needed which can stabilize the injury site and promote wound healing. Unfortunately, current corneal puncture injury models are not capable of producing irregularly shaped, large, high-speed injuries as seen on the battlefield, making relevant therapeutic development challenging. Here, we present a benchtop corneal puncture injury model for use with enucleated eyes that utilizes a high-speed solenoid device suitable for creating military-relevant injuries. We first established system baselines and ocular performance metrics, standardizing the different aspects of the benchtop model to ensure consistent results and properly account for tissue variability. The benchtop model was evaluated with corneal puncture injury objects up to 4.2 mm in diameter which generated intraocular pressure levels exceeding 1500 mmHg. Overall, the created benchtop model provides an initial platform for better characterizing corneal puncture injuries as seen in a military relevant clinical setting and a realistic approach for assessing potential therapeutics.

Blast Exposure Induces Ocular Functional Changes with Increasing Blast Over-pressures in a Rat Model.

Purpose: Blast-related brain and ocular injuries can lead to acute and chronic visual dysfunction. The chronic visual consequences of blast exposure and its progression remain unclear. The goal of this study is to analyze ocular functional response to four levels of blast exposure and identify a threshold of blast exposure leading to acute and chronic visual dysfunction. Methods: Anesthetized adult male Long-Evans rats received a single-blast exposure at a peak overpressure of 78, 117, 164 or 213 kPa, delivered by a compressed air-driven shock tube. Clinical eye examination, intraocular pressure (IOP), flash electroretinography (fERG) and spectral-domain optical coherence tomography (SD-OCT) images were assessed prior to, and at multiple time points post exposure. Results: No abnormal fERG were observed for the two lowest-level blast groups (78 kPa or 117 kPa). For the 164 kPa group, the a- and b-wave amplitudes of the fERG were decreased at 3 days postexposure (p = 0.009 for a-wave, p = 0.010 for b-wave), but recovered to baseline levels by 7 days post-exposure. The IOP was unchanged for the 117 kPa and 164 kPa groups. The 78 kPa group demonstrated a small transient increase during week one (p = 0.046). For the highest blast group (213 kPa), the IOP was significantly elevated immediately post-exposure (p = 0.0001), but recovered by 24 hr. A bimodal depression in the fERG a- and b-wave amplitudes was observed for this group: the amplitudes were depressed at day 3 post-exposure (p = 0.007 for a-wave, p = 0.012 for b-wave), and recovered by day 7 post-exposure. However, the fERG amplitudes were once again depressed at week 8 post-exposure, suggesting a chronic retinal dysfunction. All retinae appeared normal in SD-OCT images. Conclusions: Our study demonstrates that a single-blast exposure may result in acute and chronic fERG deficit, and traumatic IOP elevation. Noninvasive functional tests may hold promise for identifying individuals with a risk for developing chronic visual deficits, and indicating a time window for early clinical diagnosis, rehabilitation, and treatment.

Early Complement and Fibrinolytic Activation in a Rat Model of Blast-Induced Multi-Organ Damage.

OBJECTIVE: Blast injury is associated with multi-organ failure (MOF), causing significant morbidity and mortality in trauma patients. However, the pathogenesis of blast-induced MOF still remains obscure. In this study, we evaluate the pathophysiological changes related to blast-induced MOF in a clinically relevant rat model of blast injury. METHODS: A moderate blast overpressure was applied to induce injury in anesthetized rats. Pathological changes were evaluated by H&E staining. Complement activation, plasminogen, and myeloperoxidase levels were analyzed by complement hemolytic assay (CH50) and/or ELISA in blood samples. RESULTS: Analysis of lung, brain, and liver tissue at 24 hour after blast overpressure revealed severe injuries. The level of complement components C3 and C1q decreased in parallel with the reduction of CH50 level in injured animals at 1, 3, and 6 hours after blast. Consumption of plasminogen was also detected as early as 1 hour post-injury. Myeloperoxidase levels were elevated within 1 hour of blast injury. CONCLUSION: Our data reveal that blast injury triggers the complement and fibrinolytic systems, which likely contribute to blast-induced MOF. Conceivably, therapies that target these systems early may improve clinical outcomes in blast patients.

Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses.

The laser-induced retinal injury thresholds for repetitive-pulse exposures to 100-µs-duration pulses at a wavelength of 532 nm have been determined for exposures of up to 1000 pulses in an in vivo model. The ED50 was measured for pulse repetition frequencies of 50 and 1000 Hz. Exposures to collimated beams producing a minimal retinal beam spot and to divergent beams producing a 100-µm-diameter retinal beam spot were considered. The ED50 for a 100-µs exposure was measured to be 12.8 µJ total intraocular energy for a minimal retinal beam spot exposure and 18.1 µJ total intraocular energy for a 100-µm-diameter retinal beam spot. The threshold for exposures to N > 1 pulse was found to be the same for both pulse repetition frequencies. The variation of the ED50 with the number of pulses is described well by the probability summation model, in which each pulse is considered an independent event. This is consistent with a threshold-level damage mechanism of microcavitation for single-pulse 100-µs-duration exposures. The data support the maximum permissible exposure levels for repetitive-pulse exposure promulgated in the most recent laser safety guidelines.

Damage threshold from large retinal spot size repetitive-pulse laser exposures.

The retinal damage thresholds for large spot size, multiple-pulse exposures to a Q-switched, frequency doubled Nd:YAG laser (532 nm wavelength, 7 ns pulses) have been measured for 100 µm and 500 µm retinal irradiance diameters. The ED50, expressed as energy per pulse, varies only weakly with the number of pulses, n, for these extended spot sizes. The previously reported threshold for a multiple-pulse exposure for a 900 µm retinal spot size also shows the same weak dependence on the number of pulses. The multiple-pulse ED50 for an extended spot-size exposure does not follow the n dependence exhibited by small spot size exposures produced by a collimated beam. Curves derived by using probability-summation models provide a better fit to the data.

Near-UV/blue light-induced fluorescence in the human lens: potential interference with visual function.

Irradiation of the ocular lens of numerous species by near-UV or short-visible wavelengths induces a blue-green fluorescence, which can be a source of intraocular veiling glare. Wavelengths longer than the approximately 365-nm lens absorption peak induce progressively weaker but also progressively more red-shifted fluorescence emission. The more red-shifted emission has a higher luminous efficiency and, in fact, earlier studies in this laboratory have demonstrated that the lens fluorescence in the nonhuman primate yields an approximately constant luminous efficiency when excited by equal radiant exposures over the wavelength range from 350 to 430 nm. Now, with the recent development and projected widespread use of "blue" diode lasers, a further study extending the measurements of the induced fluorescence efficiency and of the consequent veiling glare to the human lens seemed timely. The current study quantifies the fluorescence intensity induced in the human lens, both in terms of radiance and luminance, as a function of exciting light intensity, excitation wavelength, and subject age. The spatial distribution of the emitted fluorescence is also examined. These data are shown to imply that exposure to near-UV/blue wavelength sources at "safe" exposure levels (according to existing laser safety standards) can induce a veiling glare intense enough to degrade visual performance, and that the fluorescence intensity and consequent glare disruption show little dependence on subject age.